Baylis-Hillman reaction assisted parallel synthesis of 3, 5-disubstituted isoxazoles and their in vivo bioevaluation as antithrombotic agents

The solution phase parallel synthesis involving reactions of Baylis-Hillman products of 3-substituted-5-isoxazolecarbaldehydes with nucleophiles and their in vivo antithrombotic evaluations are described along with the results of in vitro platelet aggregation inhibition assay of a few compounds. Results of the detailed evaluation of one of the compounds as an inhibitor of platelet aggregation are also presented.

Combinatorial synthesis and biological evaluation of isoxazole-based libraries as antithrombotic agents

The 3-substitutedphenyl-5-isoxazolecarboxaldehydes have been identified as activated aldehydes for the generation of isoxazole-based combinatorial libraries on solid phase through automation. Three highly functionalized isoxazole-based libraries comprising of 32, 96 and 45 compounds each have been synthesized in parallel format using Baylis Hillman reaction, Michael addition, reductive amination and alkylation reactions. With an objective of lead generation all the three libraries were evaluated for their antithrombin activity in vivo.

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

Prior Antithrombotic Use is Associated with Favorable Mortality and Functional Outcomes in Acute Ischemic Stroke

Sources of Funding The GWTG-Stroke program is currently supported in part by a charitable contribution from Bristol-Myers Squibb/Sanofi Pharmaceutical Partnership and the American Heart Association Pharmaceutical Roundtable. GWTG-Stroke has been funded in the past through support from Boehringer-Ingelheim and Merck. These funding agencies did not participate in design or analysis, manuscript preparation, or approval of this study.

Drug utilisation in a neonatal intensive care unit of a swiss university hospital

Introduction Medication errors in hospitalsmay occur at any step of the medication process including prescription, transcription, preparation and administration, and may originate with any of the actors involved. Neonatal intensive care units (NICU) take care of extremely frail patients in whom errors could have dramatic consequences. Our objective was to assess the frequency and nature of medication errors in the NICU of a university hospital in order to propose measures for improvement.Materials & Methods The design was that of an observational prospective study over 4 consecutivemonths. All patients receiving C 3drugs were included. For each patient, observations during the different stages were compiled in a computer formulary and compared with the litterature. Setting: The 11-bed NICU of our university hospital.Main outcome measures:(a) Frequency and nature of medication errors in prescription,transcription, preparation and administration.(b) Drugs affected by errors.Results 83 patients were included. 505 prescriptions and transcriptions, 447 preparations and 464 administrations were analyzed. 220 medications errors were observed: 102 (46.4%) at prescription, 25 (11.4%) at transcription, 19 (8.6%) at preparation and 73 (33.2%) at administration. Uncomplete/ambiguous orders (24; 23.5%) were the most common errors observed at prescription, followed by wrong name (21; 20.6%), wrong dose (17; 16.7%) and omission (15; 14.7%). Wrong time (33; 45.2%) and wrong administration technique (31; 42.5%) were the most important medication errors during administration. According to the ATC classification, systemic antibacterials (53; 24.1%) were the most implicated, followed by perfusion solutions (40; 18.2%), respiratory system products (30; 13.6%), and mineral supplements and antithrombotic agents (20; 9.1%).Discussions, Conclusion Proposed recommendations: ? Better teaching of neonatal prescription to medical interns;? Improved prescription form to avoid omissions and ambiguities;? Development of a neonatal drug formulary, including prescription,preparation and administration modalities to reduce errors at different stages;? Presence of a clinical pharmacist in the NICU.Disclosure of Interest None Declared

A utilização do safrol, principal componente químico do óleo de sassafráz, na síntese de substâncias bioativas na cascata do ácido araquidônico: antiinflamatórios, analgésicos e anti-trombóticos

In this paper we describe the results of a research effort developed in Laboratório de Avaliação e Síntese de Substancias Bioativas (LASSBio, UFRJ) in the utilization of Brazilian abundant natural product, safrole (1), the principal chemical constituent of Sassafras oil (Ocotea pretiosa), as an attractive synthon to access different chemical class of bioactive compounds, as prostaglandins analogues, non-steroidal antiinflammatory agents and antithrombotic compounds.

Heterofucans from Dictyota menstrualis have anticoagulant activity

Fucan is a term used to denote a family of sulfated L-fucose-rich polysaccharides which are present in the extracellular matrix of brown seaweed and in the egg jelly coat of sea urchins. Plant fucans have several biological activities, including anticoagulant and antithrombotic, related to the structural and chemical composition of polysaccharides. We have extracted sulfated polysaccharides from the brown seaweed Dictyota menstrualis by proteolytic digestion, followed by separation into 5 fractions by sequential acetone precipitation. Gel electrophoresis using 0.05 M 1,3-diaminopropane-acetate buffer, pH 9.0, stained with 0.1% toluidine blue, showed the presence of sulfated polysaccharides in all fractions. The chemical analyses demonstrated that all fractions are composed mainly of fucose, xylose, galactose, uronic acid, and sulfate. The anticoagulant activity of these heterofucans was determined by activated partial thromboplastin time (APTT) using citrate normal human plasma. Only the fucans F1.0v and F1.5v showed anticoagulant activity. To prolong the coagulation time to double the baseline value in the APTT, the required concentration of fucan F1.0v (20 µg/ml) was only 4.88-fold higher than that of the low molecular weight heparin Clexane® (4.1 µg/ml), whereas 80 µg/ml fucan 1.5 was needed to obtain the same effect. For both fucans this effect was abolished by desulfation. These polymers are composed of fucose, xylose, uronic acid, galactose, and sulfate at molar ratios of 1.0:0.8:0.7:0.8:0.4 and 1.0:0.3:0.4:1.5:1.3, respectively. This is the fist report indicating the presence of a heterofucan with higher anticoagulant activity from brown seaweed.

Antitromboottisten lääkeaineiden enantiomeerien kromatografinen fraktiointi

Suuri osa käytössä olevista lääkeaineista on kiraalisia yhdisteitä. Lääkevalmisteet sisältävät yhdisteen enantiomeerien ominaisuuksista riippuen joko yksittäistä enantiomeeria tai näiden seosta. Antitromboottisiin eli veren hyytymistä estäviin lääkeaineisiin kuuluvat varfariini ja rivaroksabaani ovat kiraalisia yhdisteitä, joiden enantiomeerien ominaisuudet poikkeavat antitromboottisen vaikutuksen voimakkuuden suhteen. Varfariinia käytetään kliinisesti enantiomeeriensa raseemisena seoksena, kun taas rivaroksabaani on käytössä lääkevalmisteena puhtaana S-enantiomeerinaan. Lääkeaineen enantiomeerien erottaminen toisistaan on tärkeää esimerkiksi enantiomeerien puhdistamiseksi, lääkevalmisteen oikean koostumuksen varmistamiseksi tai yksittäisten enantiomeerien käyttäytymisen arvioimiseksi elimistössä. Tässä kirjallisuustyössä käsiteltiin nestekromatografian käyttöä antitromboottisiin lääkeaineisiin kuuluvien antikoagulanttien enantiomeerien fraktioinnissa. Kirjallisuudesta saadun tiedon perusteella arvioitiin, millä erotusmateriaaleilla ja millaisissa koeolosuhteissa varfariinin sekä rivaroksabaanin enantiomeerit tulisi erottaa toisistaan. Varfariinin enantiomeerien erotukseen parhaiten sopivaksi erotusmateriaaliksi todettiin kirjallisuuden perusteella kiraalinen vankomysiinipohjainen stationaarifaasi (Chirobiotic V) metanolin, etikkahapon ja veden seoksen toimiessa eluenttina. Eluentin virtausnopeudella 0,3 mL/min ja pienellä injektiotilavuudella varfariinin enantiomeerit saatiin erottumaan täydellisesti ja nopeasti toisistaan. Kirjallisuuden perusteella rivaroksabaanin enantiomeerien erotuksessa erotusmateriaalina toimii parhaiten kiraalinen selluloosapohjainen stationaarifaasi (Chiralcel OD-RH), kun eluenttina käytetään n-heksaanin ja isopropanolin seosta virtausnopeudella 1 mL/min. Varfariinin ja rivaroksabaanin enantiomeerien fraktiointiin parhaiten sopivia menetelmiä voidaan käyttää eri tilanteissa, kuten lääkevalmisteiden laadunvarmistuksessa tai enantiomeerien erotuksessa niiden synteesin jälkeen.

Pharmacological actions of nobiletin in the modulation of platelet function

Background and Purpose The discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin in the modulation of platelet function. Experimental Approach The ability of nobiletin to modulate platelet function was explored by using a range of in vitro and in vivo experimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. The fibrinogen binding, α-granule secretion and calcium mobilisation assays were performed using platelet-rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletin in vivo was assessed by measuring tail bleeding time using C57BL/6 mice. Key Results Nobiletin was shown to supress a range of well-established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilisation and thrombus formation. Nobiletin was shown to extend bleeding time in mice and reduce the phosphorylation of Akt and PLCγ2 within the collagen receptor (GPVI) - stimulated pathway, in addition to increasing the levels of cGMP and phosphorylation of VASP, a protein whose activity is associated with inhibitory cyclic nucleotide signalling. Conclusions and Implications This study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore nobiletin may represent a potential antithrombotic agent of dietary origins.

Structure of thrombin complexed with selective non-electrophilic inhibitors having cyclohexyl moieties at P1

The crystal structures of five new non-electrophilic β-strand-templated thrombin active-site inhibitors have been determined bound to the enzyme. Four co-crystallize with hirugen and inhibitor isomorphously to produce thrombin-hirugen crystals (monoclinic, space group C2), while one co-crystallizes in the hexagonal system, space group P65. A 1,4-substituted cyclohexyl moiety is conserved at the P1 position of all the inhibitors, along with a fused hetero-bicyclic five- and six-membered ring that occupies the P2 site. Amino, amidino and aminoimidazole groups are attached to the cyclohexyl ring for recognition at the S1 specificity site, while benzylsulfonyl and diphenyl groups enhance the binding at the S3 subsite. The cyclohexyl groups at the P1 positions of three of the inhibitors appear to be in the energetically favored chair conformation, while the imidazole-substituted cyclohexyl rings are in a boat conformation. Somewhat unexpectedly, the two cyclohexyl-aminoimidazole groups bind differently in the specificity site; the unique binding of one is heretofore unreported. The other inhibitors generally mimic arginyl binding at S1. This group of inhibitors combines the nonelectrophilicity and selectivity of DAPA-like compounds and the more optimal binding features of the S1-S3 sites of thrombin for peptidic molecules, which results in highly potent (binding constants 12 nM-16 pM, one being 1.1 μM) and selective (ranging from 140 to 20 000 times more selective compared with trypsin) inhibitors of thrombin. The binding modes of these novel inhibitors are correlated with their binding constants, as is their selectivity, in order to provide further insight for the design of therapeutic antithrombotic agents that inhibit thrombin directly at the active site.

Trombobectomia com anel de Vollmar e utilização do cateter de Fogarty no tratamento da trombose jugular experimental em equinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Vorapaxar in the Secondary Prevention of Atherothrombotic Events

BACKGROUND Thrombin potently activates platelets through the protease-activated receptor PAR-1. Vorapaxar is a novel antiplatelet agent that selectively inhibits the cellular actions of thrombin through antagonism of PAR-1. METHODS We randomly assigned 26,449 patients who had a history of myocardial infarction, ischemic stroke, or peripheral arterial disease to receive vorapaxar (2.5 mg daily) or matching placebo and followed them for a median of 30 months. The primary efficacy end point was the composite of death from cardiovascular causes, myocardial infarction, or stroke. After 2 years, the data and safety monitoring board recommended discontinuation of the study treatment in patients with a history of stroke owing to the risk of intracranial hemorrhage. RESULTS At 3 years, the primary end point had occurred in 1028 patients (9.3%) in the vorapaxar group and in 1176 patients (10.5%) in the placebo group (hazard ratio for the vorapaxar group, 0.87; 95% confidence interval [CI], 0.80 to 0.94; P<0.001). Cardiovascular death, myocardial infarction, stroke, or recurrent ischemia leading to revascularization occurred in 1259 patients (11.2%) in the vorapaxar group and 1417 patients (12.4%) in the placebo group (hazard ratio, 0.88; 95% CI, 0.82 to 0.95; P=0.001). Moderate or severe bleeding occurred in 4.2% of patients who received vorapaxar and 2.5% of those who received placebo (hazard ratio, 1.66; 95% CI, 1.43 to 1.93; P<0.001). There was an increase in the rate of intracranial hemorrhage in the vorapaxar group (1.0%, vs. 0.5% in the placebo group; P<0.001). CONCLUSIONS Inhibition of PAR-1 with vorapaxar reduced the risk of cardiovascular death or ischemic events in patients with stable atherosclerosis who were receiving standard therapy. However, it increased the risk of moderate or severe bleeding, including intracranial hemorrhage. (Funded by Merck; TRA 2P-TIMI 50 ClinicalTrials.gov number, NCT00526474.)

[New oral anticoagulants - influence on coagulation tests].

The new oral anticoagulants (NOACs) represent alternative antithrombotic agents for prophylaxis and therapy of thromboembolic diseases. They act either by inhibition of the clotting factor Xa or IIa (thrombin). As a consequence, they influence several coagulation assays (for example prothrombin time, activated partial thromboplastin time). Because of the short half-life of these new agents, these changes show great variations in the course of 24 hours. Furthermore, there are significant differences of laboratory results depending on the used reagents. We explain the influence of apixaban, rivaroxaban (factor Xa inhibitors) and dabigatran (thrombin inhibitor) on the most commonly used coagulation assays. Besides we show that this influence depends on the way of action of the drug as well as on the principle of the coagulation assay. Being aware of this relationships helps to interpret the results of coagulation assays under influence of NOACs correctly.

Development and Characterization of Monovalent and Bivalent RNA Aptamers Targeting the Common Pathway of Coagulation

Anticoagulant agents are commonly used drugs to reduce blood coagulation in acute and chronic clinical settings. Many of these drugs target the common pathway of coagulation because it is critical for thrombin generation and disruption of this portion of the pathway has profound effects on the hemostatic process. Currently available drugs for these indications struggle with balancing desired activity with immunogenicity and poor reversibility or irreversibility in the event of hemorrhage. While improvements are being made with the current drugs, new drugs with better therapeutic indices are needed for surgical intervention and chronic indications to prevent thrombosis from occurring.

A class of therapeutics known as aptamers may be able to meet the need for safer anticoagulant agents. Aptamer are short single-stranded RNA oligonucleotides that adopt specific secondary and tertiary structures based upon their sequence. They can be generated to both enzymes and cofactors because they derive their inhibitory activity by blocking protein-protein interactions, rather than active site inhibition. They inhibit their target proteins with a high level of specificity and bind with high affinity to their target. Additionally, they can be reversed using two different antidote approaches, specific oligonucleotide antidotes, or with cationic, “universal” antidotes. The reversal of their activity is both rapid and durable.

The ability of aptamers to be generated to cofactors has been conclusively proven by generating an aptamer targeting the common pathway coagulation cofactor, Factor V (FV). We developed two aptamers with anticoagulant ability that bind to both FV and FVa, the active cofactor. Both aptamers were truncated to smaller functional sizes and had specific point mutant aptamers developed for use as controls. The anticoagulant activity of both aptamer-mutant pairs was characterized using plasma-based clotting assays and whole blood assays. The mechanism of action resulting in anticoagulant activity was assessed for one aptamer. The aptamer was found to block FVa docking to membrane surfaces, a mechanism not previously observed in any of our other anticoagulant aptamers.

To explore development of aptamers as anticoagulant agents targeting the common pathway for surgical interventions, we fused two anticoagulant aptamers targeting Factor X and prothrombin into a single molecule. The bivalent aptamer was truncated to a minimal size while maintaining robust anticoagulant activity. Characterization of the bivalent aptamer in plasma-based clotting assays indicated we had generated a very robust anticoagulant therapeutic. Furthermore, we were able to simultaneously reverse the activity of both aptamers with a single oligonucleotide antidote. This rapid and complete reversal of anticoagulant activity is not available in the antithrombotic agents currently used in surgery.